It has been known for decades that bacterial populations are heterogeneous, however, it has been extremely difficult to develop therapeutics that effectively eliminate all members of bacterial populations. Much of this difficulty lies in th fact that individual bacteria are replicating at different rates within a population, and this lead to differences in antimicrobial susceptibility. In remains unclear what drives differences in bacteria growth rates during infection, and whether the host immune response can slow the growth of subpopulations of bacteria. Recent advances in fluorescence imaging have enabled the study of heterogeneity within bacterial populations at the single cell level. Using fluorescent transcriptional reporters, we have shown that Y. pseudotuberculosis forms multiple subpopulations within a single site of bacterial replication, based on the response of individual cells to host-derived stresses. The proximity of individual bacteria to host cells drove gene expression changes, resulting in a small population of bacteria around the periphery of replicating clusters (called microcolonies) that specifically responds to host-derived stress. The impact of host stress on individual bacteria remains unclear; does this lead to the generation of a slow growing population on the periphery of microcolonies? The studies described in this proposal will utilize fluorescent reporter constructs to visualize bacterial responses to host stress over the course of infection, and determine the effects of host-derived stresses on bacterial growth rates. We hypothesize that individual bacteria around the periphery of microcolonies simultaneously respond to multiple host stresses, which results in a slower rate of bacterial replication within this subpopulation. To test this hypothesis, we will: 1) utilize stable and unstable fluorescent proteins to determine whether the stresses imparted by the host are transient, or maintained over the course of infection; 2) develop an oral feeding model of Y. pseudotuberculosis intestinal infection, to determine if spatial regulation of gene expression within intestinal tissues impacts the ability of bacteria to disseminate to deep tissues; and 3) develop a microfluidics model to live image bacterial replication during stress responses, to determine what happens to individual bacteria as they respond to single stresses or multiple stresses simultaneously. Although these studies will focus on Y. pseudotuberculosis growth, the models developed within this proposal can easily be applied to further the study of other enteric pathogens, and additional medically-relevant bacterial pathogens, to better understand the mechanisms driving the formation of slow growing bacterial populations. By gaining a better understanding of this process, we hope to provide pertinent information for the development of novel therapeutics to eliminate all members of bacterial populations, and thus clear infection.